JPH06113838A - Hydroxylase, its production, gene coding hydroxylase and monoclonal antibody specifically reacting with hydroxylase - Google Patents

Abstract

PURPOSE:To obtain a gene coding an enzyme capable of converting CMP-NeuAc to CMP-NeuGc and a monoclonal antibody capable of specifically reacting with the enzyme by isolating the enzyme. CONSTITUTION:Admixtures other than protein are removed from cytoplasmic fractions obtained by centrifuging crushed material of cell and these fractions are separated to isolate an enzyme (cytidine-phosphoric acid N- acetylhydroxylase) capable of converting CMP-NeuAc to CMP-NeuGc from soluble fraction of cell. PCR primer or DNA probe is synthesized based on amino acid sequence of this enzyme and DNA coding this enzyme is isolated. This monoclonal antibody capable of specifically reacting with cytidine- phosphoric acid N-acetylhydroxylase is produced using the purified enzyme.

Description

Detailed Description of the Invention

[0001]

The present invention relates to cytidine monophosphate N-acetylneuraminic acid hydroxylase, a method for producing the same,
Gene encoding cytidine monophosphate N-acetylneuraminic acid hydroxylase, DN containing a part of the gene and specifically hybridizing with the DNA
A, a monoclonal antibody that specifically reacts with cytidine monophosphate N-acetylneuraminic acid hydroxylase.

[0002]

2. Description of the Related Art In the sugar chains of animal cells such as mice, N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc) coexist. There is no N-glycolylneuraminic acid (NeuGc) in the chain. However, it is known that NeuGc is found in the sugar chain of certain cancer cells even in human cells.

[0003]

As shown in FIG. 1, cytidine monophosphate N-glycolylneuraminic acid (CMP-
NeuGc) is biosynthesized by hydroxylation of the N-acetyl group at the 5-position of cytidine monophosphate N-acetylneuraminic acid (CMP-NeuAc), and the existence of an enzyme involved in the biosynthesis is known. Didn't.

The object of the present invention is to purify the enzymes involved in CMP-NeuGc biosynthesis. Another object of the present invention is to clone a gene encoding the enzyme. A further object of the present invention is to produce a monoclonal antibody that specifically binds to the enzyme.

[0005]

Means and Actions for Solving the Problems
When NeuGc is produced in mouse cells, the C
An enzyme that hydroxylates the N-acetyl group at the 5-position prior to the production of MP-NeuGc (cytidine monophosphate N-acetylneuraminic acid hydroxylase, hereinafter CMP-Neu).
It was discovered that Ac hydroxylase) was expressed and that the enzyme performed the hydroxylation in one step. Then, from the soluble fraction of cells, CMP-NeuAc
The hydroxylase was purified.

The present invention is characterized in that at least a cytochrome b5 affinity column is used for purification of this enzyme. That is, in the present invention, after removing contaminants other than proteins from the soluble fraction of cells, CMP was performed using a cytochrome b5 affinity column.
It is characterized in that CMP-NeuAc hydroxylase is purified by fractionating a fraction having -NeuAc hydroxylase activity.

Further, in the present invention, purified CM
P-NeuAc hydroxylase was cleaved to determine the amino acid sequence of the fragment, and an oligonucleotide synthesized on the basis of this amino acid sequence was used as a DNA probe, and the DNA probe encoded CMP-NeuAc hydroxylase. Is cloned. Furthermore, CMP-NeuAc hydroxylase was mass-produced by the transformed cell into which this DNA was introduced, and the cloned DNA
Is also used to suppress enzyme expression by antisense technology or gene disruption technology.

Further, in the present invention, purified CMP
-NeuAc hydroxylase using the CMP-N
It is characterized in that a monoclonal antibody specifically reacting with euAc hydroxylase is produced. Then, with this monoclonal antibody, CMP-NeuAc hydroxylase existing in serum or excised tissue is detected.

By the way, by using this CMP-NeuAc hydroxylase, CMP-NeuGc can be obtained from CMP-NeuAc at a low cost.

As an example below, C from mouse hepatocytes
Purification of MP-NeuAc hydroxylase, CMP-N
Although cloning of DNA encoding euAc hydroxylase and production of a monoclonal antibody that specifically reacts with CMP-NeuAc hydroxylase are mentioned, it goes without saying that the technical scope of the present invention is not limited to this example.

[0011]

【Example】

Example 1 Purification of CMP-NeuAc hydroxylase 1-1 To 200 g of mouse liver, 4 volumes of 0.1 mM dithiothreitol (DTT) -containing 0.1 mM Tris-HCl buffer was added and homogenized under ice cooling. 4 from homogenate
A 0-60% saturated ammonium sulfate fraction was obtained, concentrated and dialyzed, and then passed through an open column of phosphocellulose to remove impurities by adsorbing the impurities on the column. The amount of protein in each fraction was measured by the absorbance at 280 nm. The enzyme activity was measured by the method shown in FIG.

That is, 0.5 μl of the enzyme fraction obtained from the cell homogenate was added to cytochrome b5 (5 μM), the component X fraction (50 μg / ml), and CMP-Neu as a substrate.
Ac (40 μM), NADH as coenzyme (0.7 m
M), 10 m containing DTT (1 mM) as an antioxidant
Add 10 μl of M Tris-HCl buffer to 30 at 37 ° C
After incubation for 120 minutes, the reaction was stopped by adding ice-cold ethanol. The cytochrome b5 and the component X fraction are essential factors or fractions for assisting the activity of this enzyme. This was centrifuged, and the supernatant was subjected to high performance liquid chromatography (HPLC) and analyzed under the following conditions.

HPLC conditions: Column: ODS-80TM (diameter 4.6 mm, length 25)
0 mm, Tosoh) Eluent: 50 mM NH4H2PO4, Flow rate: 0.5 mm / min Analytical device: Pump; SP8700 (Spectra P)
hisics) Detector; UVIDEC (JASCO Corporation) Recorder; CR5A (Shimadzu) Detection wavelength; 271 nm Quantitative method: peak area ratio method The HPLC chromatogram is shown in FIG. Two peaks were detected and their elution positions were CMP-Neu.
It coincided with the elution position of the standard products of Ac and CMP-NeuGc. Substrate CMP-NeuAc to CMP-Ne
The conversion rate to uGc is CMP-NeuAc and CMP-
It was quantified from the ratio of the peak areas of NeuGc. The formula for calculating the conversion rate is shown below.

Conversion rate (%) = (area of CMP-NeuGc) × 100 / (area of CMP-NeuAc + CMP-
Area of NeuGc) Further, one unit is 1 μmol of CMP-Neu per minute.
It was defined as the amount of enzyme that converts Ac into CMP-NeuGc. The measured values for each fraction are shown in FIG. The elution conditions of the phosphocellulose column are as follows.

Column: Cellulose phosp
hate P11 (glass column) (diameter 50 mm, length 250 mm, Whatman) eluent; (1) starting buffer (starting b)
composition) 12.5% glycerol 10 mM sodium phosphate buffer containing 0.1 mM DTT pH = 7.0 (2) Elution buffer (elution bu)
composition) 0.5M NaCl in the starting buffer
1 amount of 1 fraction; 200 ml / fr. After passing through a phosphocellulose column, both the absorbance and the conversion activity were higher in the fractions up to an elution amount of 2 liters.

1-2 Of the elution fractions of the phosphocellulose column, the fractions having an active elution amount of up to 2 liters were applied to a DEAE-Sepharose column and fractionated by the linear gradient method. The absorbance of each fraction was 280 nm. It was measured at. 0 from each fraction.
5 μl was taken to measure the conversion activity. The results are shown in Fig. 5. The elution conditions of the DEAE-Sepharose column are as follows.

Column: DEAE-Sepharose
CL-6B (diameter 50 mm, length 160 mm) Analyzer: FPLC system (Pharmacia) Detection wavelength: 280 nm Eluent; (1) Starting buffer composition 50 mM NaCl 12.5% Glycerol 10 mM Tris-HCl buffer containing DTT pH = 7.5 (2) Between the points A and B in FIG.
A linear gradient that linearly changes from M to 0.2 M was applied to elute the active fraction. At point B,
The salt concentration was rapidly raised to 1M NaCl to elute the residual protein.

FIG. 5 shows the elution profile of the DEAE Sepharose column from CMP-NeuAc to CMP-.
The conversion rate to NeuGc and the absorbance at 280 nm are shown. From the conversion rate profile, it was shown that the conversion activity was high between the elution amount of 5 liters and 6.5 liters.

1-3 Elution amount 5 eluted from DEAE-Sepharose column
Fractions with high conversion activity between liters and 6.5 liters were collected and applied to a Red-Sepharose column. Fractionation was performed by the step gradient method, the amount of protein in each fraction was quantified, and 0.5 μl was taken from each fraction to measure the conversion activity. The elution profile of the DEAE-Sepharose column is shown in FIG. The amount of protein was measured with the BCA protein assay kit. The elution conditions of the Red-Sepharose column were as follows.

Column: Red-Sepharose column (diameter 25 mm, length 60 mm) Pump: Perista pump (Ato) Eluent: At points A and B in FIG. 8, the buffer was switched as follows.

(1) Starting buffer composition 10 mM Tris-hydrochloric acid buffer containing 70 mM NaCl pH = 7.5 (2) At point A, 10 mM Tris-hydrochloric acid buffer containing 190 mM NaCl was switched to pH = 7.5.

(3) 40 μM CMP-NeuA at point B
c, 10 mM Tris-HCl buffer containing 190 mM NaCl pH = 7.5 switched.

Flow rate: 0.11 to 0.33 ml / min Volume of 1 fraction: 10 ml / fr. Injection amount: 100 ml From the elution profile shown in FIG. 6, the conversion rate of the enzyme was higher between the elution amount of 400 ml and 600 ml.

1-4 Elution amount 40 eluted from Red-Sepharose column
Fractions with high conversion activity between 0 ml and 600 ml were collected and fractionated on a cytochrome b5 affinity column. Cytochrome b5 affinity column was purified from horse serum (Yasunori Kozutsu).
mi, et. al, J .; Biochem. 110, 42
9-435 (1991)) cytochrome b5 was treated with cyanogen bromide-activated sepharose (CNBr-activate).
It was prepared by immobilizing it on d-Sepharose). The amount of protein in each fraction and the conversion activity were measured. The elution profile of the Red Sepharose column is shown in Fig. 7 (a). The amount of protein was measured with the BCA protein assay kit. The elution conditions on the cytochrome b5 affinity column were as follows.

Column: Cytochrome b5 affinity column (diameter 9 mm, length 44 mm) Eluent: 10 m containing 0.1 mM CMP-NeuAc
M Tris-HCl buffer pH = 7.5 Analytical apparatus: FPLC system (Pharmacia) Flow rate: 0.05 ml / min Volume of 1 fraction; 0.8 ml / fr. Injection amount: 0.22 ml In the elution profile shown in Fig. 7 (a), it was confirmed that the active portion was concentrated in the portion with an elution amount of 4-10 ml.

The activity peak was analyzed by polyacrylamide gel electrophoresis (SDS-PA) using a 5.15% gradient gel.
When separated by GE), it showed a single band of 65 K daltons as shown in FIG. 7 (b), showing that the enzyme was purified as a single protein, and the specific activity of the purified sample was It was 6.8 U / mg.

1-5 The isoelectric point of the purified sample was measured by the following method. The conditions are as follows.

Gel and Gel Concentration: 5% Acrylamide 2% Ampholite (pI 4 to 6.5) 12.5% Glycerol 50 μM CMP-NeuAc was prepared by polymerizing TEMED and ammonium persulfate.

Electrophoresis condition: 200V A thin strip-shaped rectangle gel was charged with the sample and subjected to isoelectric focusing, and each lane was cut out by 5 mm.
Created a fragment. For each fragment, 100 μl of eluate (0.2 M Tris-HCl buffer containing 0.1 mM CMP-NeuAc, 12.5% glycerol, pH =
7.5) was added and left overnight to elute the protein in the gel. After protein elution, each activity was measured.
From the comparison between the position of the activity peak and the migration position of the pI marker (Bio-Rad), which was run in parallel, it was concluded that the pI was between 4.65 and 5.1, as shown in FIG.

1-6 The thermostability of the enzyme was examined in the following test.

As a sample, a crudely purified fraction fractionated with a DEAE Sepharose column was used. CMP-N using 10 mM Tris-HCl buffer as a basic buffer
The inactivation inhibitory effect of euAc, CMP, NeuAc and glycerol on the enzyme activity was examined.

About 30 μU of the sample was dissolved in the basic buffer or the basic buffer in which each reagent was added to the final concentration shown in FIG. 9 to make the sample solution amount 40 μl. This was divided into two 20 μl each, and only one was heat-treated.
The heat treatment condition is 55 ° C.1 when the enzyme activity is 50% inactivated when only the basic buffer is used.
It was set to 0 minutes. The heated sample solution which had been treated at 55 ° C. for 10 minutes was ice-cooled, and 1/10 amount thereof was taken and assayed according to FIG. The assay was performed in the same manner for the sample solution that had not been heat-treated.

The stability of the enzyme against heating was expressed by the following formula.

Thermal stability (%) = Enzyme activity of sample solution after heat treatment × 100 / Enzyme activity of untreated sample solution As shown in FIG.
About 50% of the enzyme activity of the sample solution was inactivated by the treatment for a minute, but when CMP-NeuAc or glycerol was added, the inactivation of the enzyme activity was blocked, and these compounds acted to stabilize the enzyme. Although it was shown to have it, the addition of CMP alone or NeuAc alone did not prevent the inactivation of the enzyme. The degree of deactivation was dependent on the concentration of CMP-NeuAc.

Although not shown, various surfactants, sodium chloride, NADH, flavin, DTT and the like were also examined, but none of them showed enzyme deactivation inhibiting activity.

The KM value for 1-7 CMP-NeuAc was found to be that of purified enzyme 21n.
It was determined as follows using g / ml. CMP-Neu
By changing the final concentration of Ac as shown in FIG.
The assay shown in was performed. However, cytochrome b5
To save the amount of cytochrome b5 added to the normal 1
The reaction time was 10 to 30 minutes. The results are shown in Fig. 10.

When the CMP-NeuAc concentration is plotted on the horizontal axis and the initial reaction rate is plotted on the vertical axis, a normal Michaelis-Menten type saturation curve is shown. Edi Hoffsty plot (shown in box in Figure 10), K
The M value was 5.0 μM.

Example 2 Cloning of DNA encoding CMP-NeuAc hydroxylase 2-1 Purified preparation of CMP-NeuAc hydroxylase About 6 to 6
12 μg (0.1 to 0.2 nmol) was dialyzed against 0.1% sodium lauryl sulfate (SDS) to remove salts, and the mixture was applied to a protein sequencer 470A (Applied Bio Systems (ABI)) to obtain N-terminal. The 40 amino acid sequence was determined. The sequence of the determined amino acids is shown in FIG.

2-2 In order to selectively cleave the lysine residue portion of the protein,
Lysyl endopeptidase was used, which breaks down the protein into several peptides. First, about 45 μg (0.7 nmol) of a purified sample of this enzyme was treated with 8M urea to denature it, and then 5% with 0.15M Tris-HCl buffer pH 9.0.
Diluted to 0.4 μg of lysyl endopeptidase (l
Ysyl endopeptidase) was allowed to act, and the mixture was treated overnight at 37 ° C. (reaction volume 0.2 ml) to decompose this enzyme into several peptides (FIG. 12).
(A)). FIG. 12 (b) shows the results of fractionating these peptides after treatment with lysyl endopeptidase using a reverse phase column. For 14 of these peaks, A
The amino acid sequence was determined by applying a protein sequencer manufactured by BI. Two of the determined amino acid sequences are shown in FIG.
14 and FIG. 14 are shown as MZ22 and MZ28, respectively.

2-3 The nucleotide sequences predicted from the amino acid sequence of the hydroxylase are shown below the two peptides (FIGS. 13 and 1).
4). Polymerase Chain Reaction (pol
Since the DNA is amplified by using the polymerase chain reaction (hereinafter abbreviated as PCR), FIG.
Primers were synthesized for 3 and the portion with relatively low degeneracy in FIG. For the part of HZ22, primer 2 (64 mixed primers), which is a sense primer, was used in the same manner as MZ28.
Antisense primer (anti
-Primer C and primer D (sense primer) were prepared (48 types of primer C and 96 types of primer D) (FIGS. 13 and 14). Using the mouse liver cDNA as a template, the cDNAs between the primer 2 and the primer C and between the primer 2 and the primer D were amplified by the PCR method, and their nucleotide sequences were determined. The reaction conditions used for amplification are shown in FIG.

FIG. 17 shows primer 2 and primer C, and primer 2 and primer D shown in FIG.
2% agarose gel electrophoresis image of DNA amplified by the combination of As a size marker, λ-E
CoT14I (Takara) was used and electrophoresed at 120V constant voltage for 45 minutes to obtain ethidium bromide (eth
The DNA was detected by staining with an iodide bromide). Mouse brain cDNA served as a negative control. In addition, the present inventor
We have found that P-NeuAc hydroxylase is not expressed. Of the bands shown in FIG. 17, the band without an arrow also appears in the brain, or the primer D
It is also a non-specific band because it appears in the amplification using only the DNA, and the other two bands indicated by arrows are DNs encoding the target CMP-NeuAc hydroxylase.
A fragment.

Of these DNA fragments, the base sequence was determined for the larger size fragment. The nucleotide sequence was determined by the method shown in FIG.

FIG. 19 shows the nucleotide sequence (123 bp, 41 amino acids excluding the primer portion) of the DNA encoding the determined amino acids in the region between MZ22 and MZ28.
Shown in. A composite primer synthesized based on this 123 bp base sequence and poly A tail (poly A tai)
In the same manner, the C-terminal cDNA was amplified by the PCR method using the oligo dT primer that hybridized with l), and a DNA fragment encoding the C-terminal of CMP-NeuAc hydroxylase of about 700 bp was obtained.

Further, using the composite primer synthesized based on the base sequence of 123 bp obtained here and the composite primer synthesized based on the base sequence predicted from the N-terminal amino acid sequence shown in FIG. , About 1.5 Kbp of CMP-NeuAc by amplifying N-terminal cDNA by PCR method
A DNA fragment encoding the N-terminal side of hydroxylase was obtained.

Further, the DN of the region sandwiched between the start codon of DNA encoding CMP-NeuAc hydroxylase and the composite primer synthesized based on the nucleotide sequence predicted from the N-terminal amino acid sequence shown in FIG.
A was synthesized based on the determined N-terminal amino acid sequence and the optimal codon.

By these methods, the coding region of CMP-NeuAc hydroxylase (coding coding)
DNA was isolated over a total of about 2.3 Kbp.

2-4 mRNA was isolated from mouse liver according to a conventional method. A virus-derived reverse transcriptase was allowed to act on this to obtain cDNA. Furthermore, RNase H and DNA derived from Escherichia coli
Double-stranded DNA was prepared by allowing a polymerase to act. After adding EcoRI adapter to this DNA, λgt
It was incorporated into the EcoRI site of 10 vector. Using the recombinant vector thus obtained, λ phage was reconstructed to prepare a cDNA library.

The λgt10 library prepared as described above was sowed as to be about 2 × 10 ¹ per dish, and plaques were formed and transferred to a nitrocellulose membrane. Next, the 123 bp cDNA fragment obtained by PCR was labeled with ³² P using the random primer method to form a probe, and plaque hybridization according to information was performed on the nitrocellulose membrane already obtained. 2 in total
By screening 10 ⁶ phages, 3 positive clones were obtained and designated as λmZ-1, λmZ-2, and λmZ-3. Of these clones, the DNA sequence of λmZ -2 with the longest insert was determined and contained all of the expected NeuAc open reading frames.

Example 3 Production of Monoclonal Antibody Reacting specifically with CMP-NeuAc Hydroxylase 3-1 Monoclonal antibody was prepared by the method of Umeda et al.
No. 0 (1988)) was prepared as follows.

About 100 μl of the purified enzyme was directly adsorbed on the nitrocellulose membrane.

Wistar rats aged 10 to 12 weeks were used as immunized animals, and the Nembutal stock solution (50 mg / m 2) was used.
1) was intraperitoneally administered in an amount of 150 to 200 μl.

The antigen solution was homogenized into a suspension after cutting the nitrocellulose adsorbed with the protein as described above with scissors, and 0.2 ml of this was intrasplenically administered. 5
26 days after the first immunization with an antigen dose of -10 μg / rat,
Boosted twice on day 30 with immunoglobulin (IgG)
Was formed.

A hybridoma was prepared using this IgG. As myeloma cells, P3-X63-Ag
8.653 was used. The pre-washed myeloma was added to the splenocyte suspension at a ratio of 1:10 with the splenocytes, and
After collecting the cells with 100% polyethylene glycol, spread them on a 96-well plate at 200 μl / well and 5% CO ₂
Cultured at 37 ° C in an incubator. After 10 to 14 days, when colonies became visible to the naked eye, EL
The antibody was screened by ISA. By the above operation, a monoclonal antibody that specifically reacts with CMP-NeuAc hydroxylase was obtained.

On the nitrocellulose membrane, the above-mentioned antigens are in a denatured state, so that they have higher immunogenicity than usual, and when CMP-Neu is subjected to Western blotting.
A highly selective antibody that specifically reacts with Ac hydroxylase was obtained.

3-2 The full-length cDNA of this enzyme and a part thereof were incorporated into a vector and introduced into Escherichia coli to express the enzyme protein and a part thereof. Immunize animals with these as antigens and
A monoclonal antibody that specifically reacts with CMP-NeuAc hydroxylase was obtained by a method similar to.

CMP-NeuAc hydroxylases of various species can be isolated by the same method as that described in Example 1 above.

Further, the DN obtained by the above-mentioned Example 2
CM of various species by screening DNA libraries of various species using A fragment as a probe
CMP-encoding P-NeuAc hydroxylase
The gene for NeuAc hydroxylase can be cloned. Further, if the DNA obtained in this Example is used as a probe, it may be possible to easily detect cancer cells by analysis by Northern blotting. CM in cancer cells
When P-NeuAc hydroxylase is expressed, the corresponding messenger RNA is produced, whereas in normal cells, CMP-NeuAc hydroxylase is not expressed and the corresponding messenger RNA is not produced. Because.

Further, according to the present invention, CMP-NeuAc
Since the DNA encoding the hydroxylase has been cloned, the DNA used for the antisense technology and the gene disruption technology of cells can be easily prepared. Then, by using antisense technology or cell gene disruption technology, CMP-Neu is used.
Suppresses the expression of Ac hydroxylase and reduces CMP-NeuA
A sugar chain can be designed by preparing a cultured cell that does not produce c-hydroxylase.

Furthermore, by using the antibody or antiserum obtained in Example 3 above, CMP existing in the serum or excised tissue of the patient
-NeuAc hydroxylase can be detected with high sensitivity.

[0060]

As described above, C obtained by the present invention
Conventional CTP using MP-NeuAc hydroxylase
CMP-NeuGc which was synthesized from NeuGc
Can be inexpensively synthesized using CMP-NeuAc as a raw material.

Furthermore, by using the antibody or antiserum obtained by the present invention, it becomes possible to detect a predetermined cancer at an early stage.

Further, by using the DNA obtained in the present invention, it is possible to easily carry out gene diagnosis, suppress expression by antisense technology or cell gene disruption technology, and carry out sugar chain design associated therewith. Becomes

[Brief description of drawings]

FIG. 1 is a diagram showing a reaction in which CMP-NeuAc as a substrate is hydroxylated to produce CMP-NeuGc.

FIG. 2 is a diagram showing an assay method for the enzyme.

FIG. 3: Hydroxylation of CMP-NeuAc, which is a substrate, into C
It is a chromatogram of HPLC analysis at the time of producing MP-NeuGc.

FIG. 4 is a diagram showing a chromatogram when a phosphocellulose column P11 is used.

FIG. 5 is a diagram showing a chromatogram when a DEAE-Sepharose column is used.

FIG. 6 is a diagram showing a chromatogram when a Red-Sepharose column is used.

FIG. 7 (a) is a diagram showing a chromatogram when a cytochrome b5 affinity column is used, and FIG. 7 (b) is a polyacrylamide gel electrophoresis result when a cytochrome b5 affinity column is used. Is a photograph showing.

FIG. 8 is a diagram showing the conversion rate (%) of CMP-NeuAc hydroxylase and pI determined by the moving position of a standard pI marker.

FIG. 9 is a diagram showing the results of examining the thermostability of CMP-NeuAc hydroxylase.

FIG. 10 is a diagram showing the results of examining the effect of CMP-NeuAc concentration on the thermostability of CMP-NeuAc hydroxylase. In the middle of the graph is the Edy Hofsty plot.

FIG. 11 shows the N-terminal amino acid sequence of CMP-NeuAc hydroxylase and the corresponding base sequence.

FIG. 12: CMP-N by lysyl endopeptidase
It is the figure which showed the procedure (a) of the limited hydrolysis of euAc hydroxylase refined product, and the HPLC analysis result (b) of the limited hydrolysis product.

FIG. 13 shows the amino acid sequence of MZ22 and the corresponding DNA sequence.

FIG. 14 shows the amino acid sequence of MZ28 and the corresponding DNA sequence.

FIG. 15 is a diagram showing reaction conditions of PCR method.

FIG. 16 is a diagram showing the positions of PCR primers corresponding to the full length of CMP-NeuAc hydroxylase.

FIG. 17: liver and brain primer 2 and primer C,
It is a figure which shows the result of electrophoresis of the DNA fragment of the said enzyme expressed when it amplified by PCR method using the primer 2 and the primer D.

FIG. 18 is a diagram showing a procedure of sequencing.

FIG. 19 is a diagram showing the nucleotide sequences of the DNA amplified between the primer portions used in the PCR method and those primer portions.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12P 21/08 8214-4B // C12N 15/08 (C12P 21/08 C12R 1:91) (72 ) Inventor Takehiro Kawano 3-16-14-501 Tsudanuma, Narashino City, Chiba Prefecture (72) Yasunori Kotsumi 11-1 Saga Asahi-cho, Ukyo-ku, Kyoto 311 Lumiere Arashiyama

Claims (5)

[Claims] 1. Cytidine monophosphate N-acetylneuraminic acid which hydroxylates the N-acetyl group at the 5-position of cytidine monophosphate N-acetylneuraminic acid (CMP-NeuAc) to convert it into an N-glycolyl group. Hydroxylase. 2. A method for producing cytidine monophosphate N-acetylneuraminic acid hydroxylase, which comprises the steps of: 1) a step of centrifuging a cell lysate to separate a cytoplasmic fraction; ) A step of removing contaminants other than proteins from the cytoplasmic fraction, 3) a step of fractionating a fraction having a cytidine monophosphate N-acetylneuraminic acid hydroxylase activity using at least a cytochrome b5 affinity column. 3. A D containing a gene encoding cytidine monophosphate N-acetylneuraminic acid hydroxylase.
NA. 4. A DNA containing at least a part of a gene encoding cytidine monophosphate N-acetylneuraminic acid hydroxylase and specifically hybridizing with a DNA encoding cytidine monophosphate N-acetylneuraminic acid hydroxylase. Soybean DNA. 5. A monoclonal antibody which specifically reacts with cytidine monophosphate N-acetylneuraminic acid hydroxylase.